The mitochondrial permeability transition pore is a dispensable element for mitochondrial calcium efflux.

Department of Morphology, Surgery and Experimental Medicine, Section of Pathology, Oncology and Experimental Biology, Interdisciplinary Center for the Study of Inflammation (ICSI), Laboratory for Technologies of Advanced Therapies (LTTA), University of Ferrara, Ferrara, Italy.

Abstract

The mitochondrial permeability transition pore (mPTP) has long been known to have a role in mitochondrial calcium (Ca(2+)) homeostasis under pathological conditions as a mediator of the mitochondrial permeability transition and the activation of the consequent cell death mechanism. However, its role in the context of mitochondrial Ca(2+) homeostasis is not yet clear. Several studies that were based on PPIF inhibition or knock out suggested that mPTP is involved in the Ca(2+) efflux mechanism, while other observations have revealed the opposite result. The c subunit of the mitochondrial F1/FO ATP synthase has been recently found to be a fundamental component of the mPTP. In this work, we focused on the contribution of the mPTP in the Ca(2+) efflux mechanism by modulating the expression of the c subunit. We observed that forcing mPTP opening or closing did not impair mitochondrial Ca(2+) efflux. Therefore, our results strongly suggest that the mPTP does not participate in mitochondrial Ca(2+) homeostasis in a physiological context in HeLa cells.

Calcium signaling during F1/FO ATP synthase c subunit silencing in HeLa cells. The mRNA relative aboundance of ATP5G1, ATP5G2 and ATP5G3 (Ai) and the protein relative aboundance of ATP5G (Aii) after human cervical carcinoma (HeLa) cells transfection with a scrambled siRNA (siSCR) or a mix of siRNAs targeting ATP5G1, ATP5G2 and ATP5G3 (siATP5G) for 48 h (n = 3, independent experiments). Fluorescence intensity levels in the Calcein-Co2+ assay (B). HeLa cells were transfected as in (A) but in a combination with a plasmid encoding a mitochondrial red fluorescent protein (mtDsRED). These cells were monitored using fluorescence microscopy to assess the Calcein signal (n = 250, cells from 3 independent experiments). HeLa cells were transfected as in (A) but in a combination with a plasmid coding for a mitochondrial (C) (n = 30, from 6 indipendent experiments) or cytosolic (E) (n = 10, from 3 indipendent experiments) aequorin and then stimulated with 100 μM histamine (Hist). The rates of mitochondrial (D) and cytosolic (F) Ca2+ accumulation and release and a schematic representation of the meaning of the indexes are shown. The data are presented as means ± SEM; *p < 0.05,**p < 0.01.

Modulation of the mPTP activity in combination with H2O2 treatment. HeLa cells were transfected with a plasmid coding for a mitochondrial targeted aequorin, treated with 1 μM CsA for 30 min and then treated with 500 μM H2O2 for 30 min (A and B). The cells were then stimulated with 100 μM histamine (Hist) (A) (n = 10, from 3 independent experiments). Rates of mitochondrial (B) Ca2+ accumulation and release. HeLa cells were transfected with a scrambled siRNA (siSCR) or a mix of siRNAs targeting ATP5G1, ATP5G2 and ATP5G3 (siATP5G) in a combination with a plasmid coding for a mitochondrial aequorin for 48 h and treated with 500 μM H2O2 for 30 min (C and D). The cells were then stimulated with 100 μM histamine (Hist) (C). (n = 10, from 3 independent experiments). Rates of mitochondrial (D) Ca2+ accumulation and release. HeLa cells were mock transfected or transfected for 48 h with a plasmid encoding MYC-tagged ATP5G1 in a combination with a plasmid coding for a mitochondrial aequorin and treated with 500 μM H2O2 for 30 min (E and F). The cells were then stimulated with 100 μM histamine (Hist) (E) (n = 10, from 3 independent experiments). Rates of mitochondrial Ca2+ accumulation and release (F). The data are presented as means ± SEM; *p < 0.05, ***p < 0.001.

Modulation of the mPTP activity by combined CGP37157 exposure and c subunit silencing or c subunit overexpression. HeLa cells were transfected with a scrambled siRNA (siSCR) or a mix of siRNAs targeting ATP5G1, ATP5G2 and ATP5G3 (siATP5G) in a combination with a plasmid coding for a mitochondrial aequorin for 48 h and treated with 10 μM CGP37157 added 2 min before stimulation with histamine, which was done in the continuous presence of CGP37157 (A and B). The cells were then stimulated with 100 μM histamine (Hist) (A) (n = 10, from 3 independent experiments). Rates of mitochondrial Ca2+ accumulation and release (B). (C and D) HeLa cells were mock transfected or transfected for 48 h with a plasmid encoding MYC-tagged ATP5G1 in a combination with a plasmid coding for a mitochondrial aequorin and treated with 10 μM CGP37157 added 2 min before stimulation with histamine, which was done in the continuous presence of CGP37157. The cells were then stimulated with 100 μM histamine (Hist) (C) (n = 10, from 3 independent experiments). Rates of mitochondrial (D) Ca2+ accumulation and release (D). The data are presented as means ± SEM.

Modulation of the mPTP activity by CSA treatment in combination with CGP37157 treatment or MCUa overexpression. HeLa cells were transfected for 48 h with a plasmid coding for a mitochondrial aequorin and treated with 10 μM CGP37157 for 2 min, in absence or presence of 1 μM CsA for 30 min. (A and B). Ca2+ uptake was elicited by 100 μM histamine (Hist). (B) Rates of mitochondrial Ca2+ accumulation and release (n = 11, from 3 independent experiments). HeLa cells were mock transfected or transfected for 48 h with a plasmid encoding MCU-FLAG in a combination with a plasmid coding for a mitochondria-targeted aequorin in absence or presence of 1 μM CsA for 30 min (C and D). The cells were then stimulated with 100 μM histamine (Hist) (C) (n = 9, from 3 independent experiments). Rates of mitochondrial Ca2+ accumulation and release (D). The data are presented as means ± SEM; *p < 0.05, ***p < 0.001.

Modulation of the mPTP activity by c subunit silencing, c subunit overexpression and CsA treatment in permeabilized cells. (A and B) HeLa cells were transfected with a scrambled siRNA (siSCR) or a mix of siRNAs targeting ATP5G1, ATP5G2 and ATP5G3 (siATP5G) in a combination with a plasmid coding for a mitochondrial aequorin for 48 h (n = 9, from 3 indipendent experiments). (C and D) HeLa cells were mock transfected or transfected for 48 h with a plasmid encoding MYC-tagged ATP5G1 in a combination with a plasmid coding for a mitochondrial aequorin (n = 10, from 3 indipendent experiments). (E and F) HeLa cells were transfected with a plasmid coding for a mitochondria-targeted aequorin and treated with 1 μM CsA for 30 min or vehicle (n = 10 from 3 indipendent experiments). The cells were then digitonin-permeabilized and stimulated with 1 μM [Ca2+] in EGTA-buffered buffer. Calcium efflux was stimulated by perfusion with a calcium-free buffer supplemented with Ruthenium Red (RuR). Rate of mitochondrial Ca2+ release during Ca2+ deprivation and RuR perfusion (B, D and F). The data are presented as means ± SEM.

Modulation of the mPTP activity by combined CGP37157 and c subunit silencing, c subunit overexpression or CsA treatment in permeabilized cells. (A and B) HeLa cells were transfected with a scrambled siRNA (siSCR) or a mix of siRNAs targeting ATP5G1, ATP5G2 and ATP5G3 (siATP5G) in a combination with a plasmid coding for a mitochondrial aequorin for 48 h. (C and D) HeLa cells were mock transfected or transfected for 48 h with a plasmid encoding MYC-tagged ATP5G1 in a combination with a plasmid coding for a mitochondrial aequorin (n = 12, from 3 indipendent experiments). (E and F) HeLa cells were transfected with a plasmid coding for a mitochondria-targeted aequorin and treated with 1 μM CsA for 30 min or Vehicle (n = 9, from 3 indipendent experiments). The cells were then digitonin-permeabilized and stimulated with 0.5 μM [Ca2+] in EGTA-buffered buffer and in presence of CGP37157 10 μM. Calcium efflux was stimulated by 2 μM Ruthenium Red (RuR). Rate of mitochondrial Ca2+ release during RuR perfusion (B, D and F). The data are presented as means ± SEM.